This proposed study involves two homologous proteinase inhibitors, one directed toward carboxypeptidases and the other relatively specific for chymotrypsin. Reported characteristics of the carboxypeptidase inhibitor from potatoes are amino acid sequence, KI values, and a tentative reactive site structure. Further characterization of this inhibitor and its interaction with carboxypeptidases will include pairing the disulfide bonds, preparation of crystals suitable for X-ray diffraction, and utilizing a derivative of the inhibitor as an affinity label to identify a region in carboxypeptidase A in contact with inhibitor in the complex. Also, the uniquely tight binding between the inhibitor and carboxypeptidases will be exploited in preparation of target enzymes by affinity chromatography with particular emphasis on carboxypeptidase G1, a folate antagonist under clinical investigation in the treatment of leukemia. The chymotrypsin inhibitor has recently been purified in this laboratory and partially characterized. Preliminary sequence analysis and immunological studies demonstrate that this inhibitor is homologous to the carboxypeptidase inhibitor, suggesting common ancestral origin. Complete characterization of the chymotrypsin inhibitor, including determination of amino acid sequence, paring of disulfide bonds, location of the reactive site, and preparation of suitable crystals, is proposed. These data should provide a basis for comparison with properties of the carboxypeptidase inhibitor greatly increasing our understanding of the mechanism of action and of evolution of proteinase inhibitors.